In this FY, we accomplished and advanced several important projects. They are (1) NO coordination with heme species, (2) Identifications of unique properties of parasite-derived intracellular membranes, (3) study of time-dependent protein trafficking of tetracysteine-tagged parasite KAHRP protein, (4) initiation of new project studying gametocyte metabolism and drug resistance relationship. (1) In this FY, we expanded previous project studying NO production inside parasites. We found that products of two putative parasite genes for NO-producing enzymes are expressed inside parasite. Using resonance Raman spectroscopy, we also found that NO produced in situ interact with heme species and affect equilibrium of crystallization of heme into hemozoin, which directly relates to toxicity of heme against parasites. See the 'research advance'section for more information. This work has been recently published in the Journal of Experimental Parasitology (Early electronic publication). (2) We continued studying parasite-derived membranes with unique biophysical properties. During invasion process, erythrocyte membrane-derived parasitophorous vacuole (PVM) surrounds invading parasite. PVM continuously increase in its size as parasites mature. In addition, new membranes emerge from PVM and form MC. This structure is thought to play crucial roles in trafficking and delivery processes for parasite-derived proteins. However, the source of lipid molecules enough to support the size increase in the new membrane structure is not known. A key to solve this question is cholesterol content as parasite does not have a cholesterol synthesis mechanism. In addition, cholesterol is one of the most important molecules for membrane biophysical properties. The potentially unique membrane properties of PVM and MC could provide important insights for their functions responsible for efficient trafficking and delivery of parasite proteins to host erythrocyte membrane. In previous FY, we identified unique biophysical properties of PVM and MC using Fluorescence Lifetime Microscopy (FLIM) and fluorophores (Di4-ANEPPDHQ) sensitive to cholesterol enriched membrane domains. The data suggested that these membranes have cholesterol level between host erythrocyte membrane and parasite membrane. The data suggested that membrane exchange or transfer occurred during parasite invasion and intraerythrocytic stages. In this FY year, we identified the lipid exchange/transfer between parasite and host erythrocyte membrane. The mixture of Di4-ANEPPDHQ-labelled erythrocytes and non-labeled matured parasites resulted in fluorescently positive PVMs and parasites in the ring stage in the next cycle. This fluorescence expanded throughout the parasite and PVM as it developed to matured stage, but fluorescence intensity at host membrane decreased at later stages, showing that lipid/membrane was transferred from host erythrocyte membrane to parasites during invasion process as well as following intraerythrocytic stages. In contrast, non-labeled erythrocyte mixed with labeled mature parasite did not show fluorescence from host membrane, suggesting the lipid/membrane transfer seems to be directional. On the other hand, non-parasitized erythrocytes labeled with fluorescent cholesterol analogue resulted in labeled new ring and mature parasites but host erythrocytes did not show a significant decrease in fluorescence intensity. These results suggest that lipid transfer mechanism may not be the same between cholesterol and non-sterol lipids such as phospholipids. The manuscript for these data is now under preparation. (3) In previous FY, we established protocol and methods for studying parasite protein trafficking using tetracysteine (TC) tag. we successfully engineered KAHRP-GFP-TC (control) and KAHRP-TC constructs. ReAsH-TC labeling of KAHRP-GFP-TC fusion proteins confirmed expression of TC tag and a comparable fluorescence pattern of protein distribution as GFP fluorescence, showing the capability of TC tag to be used for study of protein trafficking in a live condition. In this year, we selectively labeled old and new proteins with biarsenical fluorophores with two different colors in live condition. We quantified the time required for new protein reach MC from parasite body as well as for the protein to reach host erythrocyte membrane. Our results suggested that 5 hours was required for newly synthesized proteins appear in MC and 9 hours for host erythrocyte membrane appearance. We also found not all proteins were delivered to erythrocyte cytoplasm, but remained inside parasite. These results show that the tetracysteine technology can provide information on a new dynamic aspects of protein trafficking/delivery mechanism which is not otherwise possible with the common GFP-labeling. (4) In this year, we applied our expertise and knowledge on lipid membrane and microscopy technique and initiated a new project studying gametocyte metabolism related to drug resistance. During intraerythrocytic stages of P. falciparum, some parasites start forming gametocyte under the stress such as reductions in energy sources. Gametocytes develop through 5 different stages and eventually become gametes. However, physiological and molecular events for gametocyte metabolism are largely unknown. One physical characteristic is that gametocyte produce a large amount of internal membranes. We also confirmed this using lipid analogue fluorescent dye, Di-4 ANEPPDHQ. Majority of new membranes were produced in erythrocyte cytoplasm as a part of parasitophorous vacuole (PVM) or extension of PVM. However, labeling with Filipin III, a cholesterol-binding fluorophore, did not show any increase in cholesterol in gametocytes, suggesting only the amount of phospholipids increased. We also found in the experiment with reactive oxygen species indicator that gametocytes have significantly lower superoxide level as compare to erythrocytic stages. This was an interesting observation because it suggested that digestions of amino acids from erythrocyte hemoglobin may have decreased and established the new energy source/acquisition pathway. We are currently investigating this possibility by biochemical assay as well as fluorescence indicators for various intracellular species. On the other hand, gametocytes are known to be resistant against many of antimalarial drugs, including chloroquine. Since no known mechanism exist to explain the resistance, we start studying the expression level of PfCRT, a parasite-induced membrane protein whose mutation is responsible for the chloroquine resistance in intraerythrocytic stages. Preliminary result indicated that gametocyte did not show any reactivity against PfCRT antibody in later than stage II. Since wild-type PfCRT is essential for normal parasite growth in asexual stages, no PfCRT expression by IFA suggested that altered food vacuole function in gametocyte. Additional experiments are being performed to answer how PfCRT expression are controlled in gametocyte stages, and to study the general gametocyte energy metabolism. Collaborative Projects (Jichi Med school): We start observing MC structure by scanning electron microscope to study parasite-derived membrane system essential for protein trafficking across the host erythrocyte cytoplasm. By removing the top half membrane of substrate attached erythrocyte, we directly observed inside surface of erythrocyte membrane. Preliminary results showed that MCs were associated with erythrocyte membrane and they are connected physically by tethering membranous structure. This structure was never been reported although it was suggested. This tether sructure may provide important means for protein transport from MC to host erythrocyte membrane.